Laminopathic mutations interfere with the assembly, localization, and dynamics of nuclear lamins

Lamins are nuclear intermediate filament proteins and the major building blocks of the nuclear lamina. Besides providing nuclear shape and mechanical stability, lamins are required for chromatin organization, transcription regulation, DNA replication, nuclear assembly, nuclear positioning, and apoptosis. Mutations in human lamins cause many different heritable diseases, affecting various tissues and causing early aging. Although many of these mutations result in nuclear deformation, their effects on lamin filament assembly are unknown. Caenorhabditis elegans has a single evolutionarily conserved lamin protein, which can form stable 10-nm-thick filaments in vitro. To gain insight into the molecular basis of lamin filament assembly and the effects of laminopathic mutations on this process, we investigated mutations in conserved residues of the rod and tail domains that are known to cause various laminopathies in human. We show that 8 of 14 mutant lamins present WT-like assembly into filaments or paracrystals, whereas 6 mutants show assembly defects. Correspondingly, expressing these mutants in transgenic animals shows abnormal distribution of Ce-lamin, abnormal nuclear shape or change in lamin mobility. These findings help in understanding the role of individual residues and domains in laminopathy pathology and, eventually, promote the development of therapeutic interventions

The contribution of intracellular calcium stores to mEPSCs recorded in layer II neurones of rat barrel cortex

Loading slices of rat barrel cortex with 50 μm BAPTA-AM while recording from pyramidal cells in layer II induces a marked reduction in both the frequency and amplitudes of mEPSCs. These changes are due to a presynaptic action. Blocking the refilling of Ca2+ stores with 20 μm cyclopiazonic acid (CPA), a SERCA pump inhibitor, in conjunction with neuronal depolarisation to activate Ca2+ stores, results in a similar reduction of mEPSCs to that observed with BAPTA-AM, indicating that the source for intracellular Ca2+ is the endoplasmic reticulum. Block or activation of ryanodine receptors by 20 μm ryanodine or 10 mm caffeine, respectively, shows that a significant proportion of mEPSCs are caused by Ca2+ release from ryanodine stores. Blocking IP3 receptors with 14 μm 2-aminoethoxydiphenylborane (2APB) also reduces the frequency and amplitude of mEPSCs, indicating the involvement of IP3 stores in the generation of mEPSCs. Activation of group I metabotropic receptors with 20 μm (RS)-3,5-dihydroxyphenylglycine (DHPG) results in a significant increase in the frequency of mEPSCs, further supporting the role of IP3 receptors and indicating a role of group I metabotropic receptors in causing transmitter release. Statistical evidence is presented for Ca2+-induced Ca2+ release (CICR) from ryanodine stores after the spontaneous opening of IP3 stores

Matefin/SUN-1 is a nuclear envelope receptor for CED-4 during Caenorhabditis elegans apoptosis

In Caenorhabditis elegans, the antiapoptotic protein CED-9 is localized at the mitochondria, where it binds the proapoptotic protein CED-4. Induction of apoptosis begins when the proapoptotic protein EGL-1 is expressed and binds CED-9. The binding of EGL-1 to CED-9 releases CED-4 from CED-9 and causes the activation of the caspase CED-3. Upon its release from CED-9, CED-4 rapidly translocates to the nuclear envelope (NE) in a CED-3-independent manner. However, the identity of the NE receptor for CED-4 and its possible role in the execution of apoptosis has remained unknown. Here, we show that the inner nuclear membrane SUN-domain protein matefin/SUN-1 is the NE receptor for CED-4. Our data demonstrate that matefin/SUN-1 binds CED-4 and is specifically required for CED-4 translocation and maintenance at the NE. The role of matefin/SUN-1 in the execution of apoptosis is further suggested by the significant reduction in the number of apoptotic cells in the organism after matefin/SUN-1 down-regulation by RNAi. The finding that matefin/SUN-1 is required for the execution of apoptosis adds an important link between cytoplasmic and nuclear apoptotic events

Laminopathic mutations interfere with the assembly, localization, and dynamics of nuclear lamins

Lamins are nuclear intermediate filament proteins and the major building blocks of the nuclear lamina. Besides providing nuclear shape and mechanical stability, lamins are required for chromatin organization, transcription regulation, DNA replication, nuclear assembly, nuclear positioning, and apoptosis. Mutations in human lamins cause many different heritable diseases, affecting various tissues and causing early aging. Although many of these mutations result in nuclear deformation, their effects on lamin filament assembly are unknown. Caenorhabditis elegans has a single evolutionarily conserved lamin protein, which can form stable 10-nm-thick filaments in vitro. To gain insight into the molecular basis of lamin filament assembly and the effects of laminopathic mutations on this process, we investigated mutations in conserved residues of the rod and tail domains that are known to cause various laminopathies in human. We show that 8 of 14 mutant lamins present WT-like assembly into filaments or paracrystals, whereas 6 mutants show assembly defects. Correspondingly, expressing these mutants in transgenic animals shows abnormal distribution of Ce-lamin, abnormal nuclear shape or change in lamin mobility. These findings help in understanding the role of individual residues and domains in laminopathy pathology and, eventually, promote the development of therapeutic interventions

Suppression of Arabidopsis vesicle-SNARE expression inhibited fusion of H(2)O(2)-containing vesicles with tonoplast and increased salt tolerance

Intracellular vesicle trafficking performs essential functions in eukaryotic cells, such as membrane trafficking and delivery of molecules to their destinations. A major endocytotic route in plants is vesicle trafficking to the vacuole that plays an important role in plant salt tolerance. The final step in this pathway is mediated by the AtVAMP7C family of vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptors (v-SNAREs) that carry out the vesicle fusion with the tonoplast. Exposure to high-salt conditions causes immediate ionic and osmotic stresses, followed by production of reactive oxygen species. Here, we show that the reactive oxygen species are produced intracellularly, in endosomes that were targeted to the central vacuole. Suppression of the AtVAMP7C genes expression by antisense AtVAMP711 gene or in mutants of this family inhibited fusion of H(2)O(2)-containing vesicles with the tonoplast, which resulted in formation of H(2)O(2)-containing megavesicles that remained in the cytoplasm. The antisense and mutant plants exhibited improved vacuolar functions, such as maintenance of ΔpH, reduced release of calcium from the vacuole, and greatly improved plant salt tolerance. The antisense plants exhibited increased calcium-dependent protein kinase activity upon salt stress. Improved vacuolar ATPase activity during oxidative stress also was observed in a yeast system, in a ΔVamp7 knockout strain. Interestingly, a microarray-based analysis of the AtVAMP7C genes showed a strong down-regulation of most genes in wild-type roots during salt stress, suggesting an evolutionary molecular adaptation of the vacuolar trafficking

Cholesterol-sensitive Modulation of Transcytosis

Cholesterol-rich membrane domains (e.g., lipid rafts) are thought to act as molecular sorting machines, capable of coordinating the organization of signal transduction pathways within limited regions of the plasma membrane and organelles. The significance of these domains in polarized postendocytic sorting is currently not understood. We show that dimeric IgA stimulates the incorporation of its receptor into cholesterol-sensitive detergent-resistant membranes confined to the basolateral surface/basolateral endosomes. A fraction of human transferrin receptor was also found in basolateral detergent-resistant membranes. Disrupting these membrane domains by cholesterol depletion (using methyl-β-cyclodextrin) before ligand-receptor internalization caused depolarization of traffic from endosomes, suggesting that cholesterol in basolateral lipid rafts plays a role in polarized sorting after endocytosis. In contrast, cholesterol depletion performed after ligand internalization stimulated cargo transcytosis. It also stimulated caveolin-1 phosphorylation on tyrosine 14 and the appearance of the activated protein in dimeric IgA-containing apical organelles. We propose that cholesterol depletion stimulates the coupling of transcytotic and caveolin-1 signaling pathways, consequently prompting the membranes to shuttle from endosomes to the plasma membrane. This process may represent a unique compensatory mechanism required to maintain cholesterol balance on the cell surface of polarized epithelia